This invention relates to a novel neutral protease produced by aerobically culturing a strain belonging to Bacillus polymyxa, preferably a strain of Bacillus polymyxa deposited at the American Type Culture Collection, Rockville, Md. under accession number ATCC 21993, and at the Fermentation Research Institute, Agency of Industry and Technology, Chiba City, Japan under accession number FERM-P-No. 412, on a culture medium containing a suitable carbon source and nitrogen source.
This novel neutral protease of the present invention is useful for animal tissue and cell culture, particularly in dispersing animal tissues, in dispersing animal tissue culture cells, and in suspension culture.
Heretofore, a method of tissue and cell culture has been developed in the field of research of the basic science and has been applied to the production of virus vaccine. It is considered that such method will become one of the most important techniques for the biological industry in future.
In the process of tissue and cell culture, it is a basic technique "to disperse tissue cells".
Prior to cultivating animal cells, the animal tissue should preferably be dispersed. After placing cells in a culture vessel, the tissue culture cells attached to the surface of the vessel are removed from the surface and dispersed to obtain a cell suspension. In order to cultivate on a large scale, it is convenient to cultivate animal tissue in a dispersed state, that is, to use a suspension culture process.
Heretofore in order to disperse tissue and cells, it has been attempted to add an enzyme or a chelating agent to a solvent for dispersion, or to mechanically disperse by means of scissors, spatulas, pipettes or the like. Rous and Jones used trypsin to disperse spleen tissue, tumor-tissue and the like of a fowl and a mammal. Medawar digested the heart tissue of a chicken embryo and human skin tissue by a trypsin solution, and successfully cultivated cells from the digested material. Dulbecco prepared a cell suspension by using a salty solution of trypsin in the process of monolayer culture of chicken embryo cells, and this culture was applied to his experiments on virus plaque formation.
Among conventional methods, Rappaport's and Bodian's methods are typical ones. Both methods use trypsin, but the former produces a cell suspension in a short time by using a relatively high temperature of 37.degree.C to highly activate an enzyme which acts on the tissue employed, while the latter produces a cell suspension over a long time by using a low temperature (4.degree. to 6.degree.C) while stirring a solution containing tissue and an enzyme by means of a magnetic stirrer. Both methods provide cell suspensions which can be used for tissue culture.
Thus, trypsin was the main enzyme used for dispersing tissue and cells, but Rinaldini used pancreatin, elastase, papain and hyaluronidase in addition to trypsin in order to disperse the heart tissue of a fowl embryo, and found that elastase was useful in respect to digesting intercellular substances. Hinz et al. used collagenase to culture human, pig and rabbit lungs. Hayashi et al made a comparative test of trypsin, pancreatin and the other various enzyme-containing compounds to digest human amnoin tissue and prepared suspended cells in a high yield by action of pancreatin for a short time. Gwatkin et al. used "Pronase" (trademark), which is a protease produced by Streptomyces, to disperse mouse embryo tissue fragments, and obtained its monolayer culture.
Various enzymes and chelating agents have been studied for use in selectively cultivating a specific group of cells among various cells present in a tissue. Rappaport et al. used boron tetraphenylate, which is a chelating agent for potassium, to disperse the liver of a mouse thereby obtaining hepatic parenchymal cells. Lasfarques prepared a group of epithelial cells by treating the mammary gland tissue of a mouse with bacterial collagenase for a long time thereby damaging cells of connective tissue.
However the above mentioned enzymes, except for "Pronase" and collagenase, are all derived from animal sources. Consequently, such enzymes are restricted in respect to their sources and it is complicated to prepare them. Therefore they are rather expensive. In addition to the above general disadvantages, trypsin has many other defects in that it is inhibited by serum; that its optimum pH range of activity is relatively narrow (pH 7.2 to 7.4); that it is rapidly inactivated by incubation; that it tends to damage the cells on a certain concentration of trypsin; and accordingly that very careful operation is required. Animal tissues to which collagenase is applied are restricted since the collagenase works only with tissues containing collagen. There is a risk that crude enzymes derived from an animal source include mycoplasm. "Pronase" has disadvantages in that it is often heterogenous in quality and accordingly that reproducibility is unfavorable. According to Gwatkin's operating conditions, cells are highly apt to be damaged. In view of the above mentioned disadvantages in the prior art, an enzyme which has none of the above disadvantages and which can be easily handled, is desired.